Southern pines (Pinus) (i.e., loblolly pine, slash pine, etc.) and Douglas-fir (Pseudotsuga menziesii) are among the most important commercial species of North American timber trees. Due to the voracious demands of the lumber and paper industries, literally billions of these trees have been harvested and replanted since the early 1940's, when the first serious private reforestation efforts were initiated.
As is true with most crops, trees have been selected and manipulated by man in order to promote certain desired characteristics; such as rapid growth, resistance to insects and disease, straightness of bole, fiber length, wood density, and so on. Currently, in both the southern pine and Douglas-fir regions the majority of the seed utilized in reforestation efforts is produced from selected trees grown in seed orchards.
Although these seed orchards contain superior trees, it is extremely difficult to strictly control the pollination of the trees. Often the seed trees are fertilized by wild pollen from unselected trees. This problem can result in sexually reproduced progeny with less genetic gain potential than could have been obtained if the seed orchard trees had been fertilized by pollen from other genetically superior trees.
One method traditionally used by geneticists to solve this problem of wild pollination generating inferior genotypes is to reproduce the plant in the laboratory via tissue culture. However, forest tree species have proven difficult to reproduce in this manner. It was not until the late 1970's that Douglas-fir and loblolly pine were successfully reproduced via tissue culture. An excellent history of the development of this type of reproduction (as well as a method for reproducing coniferous plants by somatic embryogenesis) is provided by U.S. Pat. No. 4,957,866 to Gupta et al., which is hereby incorporated by reference.
Despite recent advances in procedures for laboratory reproduction of coniferous plants, a major problem remains for those researchers attempting to produce isogenic (single genotype) cell lines. Multiple zygotic embryos may form in conifer seeds due to polyembryony (Singh, 1978). Cleavage polyembryony is the division or cleavage of early stage embryos into four embryos. Cleavage embryos are genetically identical to the zygotic embryo from which they are derived. The other form of polyembryony, simple polyembryony, is due to multiple fertilization events within an individual conifer seed. It is very common for conifer seeds to have more than one egg cell per seed (Allen and Owens, 1972). Therefore, the potential exists for simple polyembryony in conifer seeds. During pollination it is also common for several male pollen grains (each is genetically different) to make their way into the seed. As a result, multiple fertilization events can occur within an individual seed. Unlike the cleavage embryos, embryos derived from different fertilization events are genetically different from each other.
As an example, if a single conifer seed contains three egg cells that are fertilized via pollen from different trees (FIG. 1) and the resulting three zygotic embryos undergo cleavage (where each divides into four embryos) (FIG. 2), then the seed would contain 12 developing embryos with three different genotypes (FIG. 3). In most conifers, as the seed matures, one embryo dominates and the other embryos fail to develop fully and eventually lose viability.
With several species of conifers, embryogenic cell cultures which can regenerate plants via somatic embryogenesis are established by culturing the entire seed (with the seed coat removed) which contains the developing immature embryos (Gupta and Durzan, 1987; Becwar et al., 1988; Finer et al., 1989; Becwar et al., 1990). The optimum stage for initiating the cultures from pine seeds is when the multiple embryos, due to simple and cleavage polyembryony, are still viable (Becwar et al., 1990; Finer et al., 1989). It is possible to initiate embryogenic cultures from more than one of the multiple zygotic embryos.
Embryogenic cultures which originate from seeds with multiple zygotic embryos, therefore, may be genetic mosaics (i.e., cultures with multiple genotypes). The possibility of having embryogenic cultures which are multigenic poses a serious limitation to the application of somatic embryogenesis to clonal forestry. In order to regenerate clonal planting stock from embryogenic cultures, it will be necessary to have isogenic cell lines. This will ensure that all of the plants regenerated from one cell line are genetically equivalent.
A common assumption (or misconception) is that explant tissues used for initiation of embryogenic conifer cultures are isogenic. In fact, for the reasons mentioned above, this may not be the case when cultures are initiated from immature conifer seeds which contain the polyembryonic tissues.
The most obvious way that the problem can be alleviated is to start with explant tissue that is known to be of a single genotype. For instance, by starting with mature zygotic embryos (since in most conifers the secondary zygotic embryos quickly loose viability as the dominant embryo matures) or isolated parts of mature zygotic embryos of conifers, one can obtain an embryogenic culture derived from solely one genotype. It is possible to do so routinely with conifers in the Picea (spruce) genus (Attree et al., 1991), but it has been much more difficult to do with conifers in the Pinus (pine) genus. Only one report (Gupta and Durzan, 1986) has claimed to initiate embryogenic cultures from mature zygotic embryos of pines, and this work has not been duplicated or repeated by other workers.
Therefore, to initiate embryogenic cultures capable of regenerating plants in several genera of conifers [including the economically important genera Pinus, Pseudotsuga (Douglas-fir), and Larix (Larch)], the only alternative is to start with zygotic embryos derived from immature seeds. One way to do so is to isolate the dominant immature zygotic embryo from the seed and culture it individually. This has worked in several species (e.g.; spruce, pine, and larch), but it is exceedingly tedious and culture initiation frequencies are lower than when the entire seed is cultured (Finer et al., 1989). Furthermore, it is difficult to ensure that one is only getting a single isolated zygotic embryo due to polyembryony and the small size of the embryos within the seed.
An alternative to isolating individual immature zygotic embryos has worked with several conifer species. With this technique, the immature seed (with the seed coat, nucellus, and megaspore wall tissues removed) which contains the developing zygotic embryos is cultured. Thus, the zygotic embryos are cultured intact within the surrounding nutritive tissue of the seed (the megagametophyte). This technique was first used with Radiata pine and since has proven effective on numerous other species (Gupta and Durzan, 1987; Becwar et al., 1988; Finer et al., 1989; Becwar et al., 1990; Norgaard and Krogstrup, 1991). Using this technique, zygotic embryos and embryogenic tissue are extruded out of the megagametophyte onto the culture medium. The extruded tissue is comprised of individual embryos (zygotic and/or somatic embryos) and individual or aggregates of suspensor cells. The embryogenic tissue proliferates from the zygotic embryos, and by transfer to an appropriate medium, further development of the somatic embryos can be induced and plants regenerated.
Other researchers have used a slight modification of the above technique where the culture is of a section of the megagametophyte (usually a transverse section) which contains the early stage zygotic polyembryonic tissue. This has proven effective for initiating embryogenic cultures of Douglas-fir (Durzan and Gupta, 1987), Abies alba (Schuller et al., 1989), and Larix species (Klimaszewska, 1989).
However, the above methods of initiating embryogenic cultures in conifers by culturing seed tissue or sections of seed tissue which contains multiple and genetically different zygotic embryos have a major limitation when used for clonal forestry. Namely, due to simple polyembryony, the extruded embryogenic tissue may be genetically heterogeneous--a genetic mosaic. Thus, somatic embryos and plants regenerated from such multigenic cultures may be genetically heterogeneous. This can cause great problems for genetic researchers attempting to produce pure genetic strains of superior stock via somatic embryogenesis for the purpose of cloning.
Therefore, it is the object of this invention to provide a process for producing isogenic cell lines of embryogenic conifer cultures. Other objects, features, and advantages will be evident from the following disclosure.